The allelic heterogeneity of BRCA1, BRCA2, and ATM mutations represents a considerable challenge for developing a diagnostic test to identify quickly, efficiently, and inexpensively all possible mutations in these large genes. An attractive option is the use of "DNA chips", high density oligonucleotide arrays synthesized on a silicon surface. In collaboration with Affymetrix, we are investigating the application of this method to BRCA1, BRCA2, and ATM mutation detection. Over 175,000 oligonucleotides were synthesized on DNA chips tailored for each gene to represent all possible nucleotide substitutions, single nucleotide insertions, (1-2)-bp deletions, and recurring mutations. Two-color competitive cohybridization experiments were performed on samples containing known mutations as well as an initial blinded scan of 50 samples which found 19/20 BRCA1 mutations present. Blinded studies on the ATM gene found 13/14 distinct heterozygous and 7/7 distinct heterozygous mutations present in 22 genome DNA samples. We are also investigating the use of modified nucleoside triphosphates to diminish the sequence-dependent hybridization efficiency of the chip assays. Inclusion of 5-methyluridine 5'phosphates (instead of rU) into BRCA2 and ATM chip analyses enhanced target hybridization while maintaining sensitivity toward single nucleotide mismatches.